With improved financial and technical supports, many malaria endemic nations are once again considering malaria elimination as their national goal of malaria control. To achieve this ambitious goal, one of the greatest challenges is development of blood stage vaccines. Antigenic variation of PfEMP1 encoded by a 60-member var gene family and clonally expressed on the surface of Plasmodium falciparum-infected red blood cells is a critical virulence factor for malaria. Despite its central role in malaria pathogenesis during the asexual blood stage, the mechanism of mutually exclusive expression of var genes is still poorly understood, and this knowledge gap severely hinders the development of novel tools to kill blood stage parasites. This project, built on our recent success in creating a P. falciparum line with expression of all the var genes, aims to determine the molecular regulatory network and establish the hierarchy of var gene regulation mechanisms employing a set of recently developed genetic and epigenetic tools and resources. A comprehensive picture of var gene regulatory factors generated from this study will enable a better understanding of antigenic variation of the var gene family in immune evasion of malaria parasites. This information will also provide new targets for therapeutics to block PfEMP1-mediated immune evasion or complications of severe malaria, or enhance the ability of vaccines to maximize PfEMP1 expression for immune response.